The present invention deals generally with electroporation, specifically the concept of spatially targeted entry of genes, drugs, chemicals and other substances into cells with a microelectronic electroporation array.
Gene therapy is an experimental method that attempts to replace, manipulate, or supplement nonfunctional or malfunctioning genes in cells with other genes to cure disease. The disease is generally one in which the patient's version of the gene is significantly different from that found the general population. As a preliminary step in studying a candidate gene to be used for therapy, that gene may be inserted into cells of the type involved in the disease. If the candidate gene alleviates the cellular component of the disease it may be studied further in vivo in an animal model of the disease. In some cases it is not known what the exact difference in the disease gene is or even which gene within a chromosome is related to the disease. In this case, one could try to carefully modify the nucleotide base pairs in the gene, testing them in cells one by one until the cause of the disease is found. The problem is with the magnitude of the search: typically there are 1000 to 100,000 base pairs within a given gene. Another problem becoming more evident is the fact that most diseases are multifactorial or result from a number of gene “defects”. Thus, one would have to study combinatorial nucleotide changes—theoretically this can become an astronomical number. What is clearly needed is a method to study the treatment of cells with large numbers of genes in a parallel or combinatorial manner.
The traditional method of study has been to serially introduce genes into cells. A separate experiment is required to study each new candidate gene. In addition, most current techniques for inserting genes into cells to study their benefit or detriment do not lend themselves to rapid parallel study. These gene insertion methods include, viral transfection, chemical weakening of the cell membrane and microinjection of the genes into individual cells. Electroporation is another method that is used to serially study gene effects.
Electroporation is the creation of nano-scale transient pores in the cell membrane with a relatively high electrical potential gradient. There are generally three existing methods for electroporating. The first is to use a chamber, or “well”, with a pair of plate electrodes. A solution containing the cells to be electroporated is placed into the well and a voltage pulse is given across the electrode pair (Neumann, et al., 1982). The second method is to use a probe with a pair or more of electrodes at the tip that can be placed on the surface, subcutaneous or deep within a surgical field to electroporate cells in vivo, or in a patient (U.S. Pat. No. 6,451,002). A third method is for cells to be cultured on the surface of an electrode (Lin and Huang. 2001, Lin et al., 2001, Huang and Rubinsky. 1999) or brought in close approximation (Olofsson et al., 2003) to an electrode and electroporated in small groups or one at a time. The problem with the prior art in this field is the lack of a high-density electrode array for electroporating, a method to hold cells safely in contact with the array and a method to put a variety of genes in contact with the cells for electroporating. Our invention is a microelectronic electroporation array with over 3000 independent electrodes. This will allow for parallel study of the cellular effect of numerous genes, drugs or other substances.
One alternative to our invention is to use a multi-well plate, a disposable cell culture container with a number of separate “wells”, 96 is a common number, to hold cell suspensions or solutions. Each well can be filled with cells in suspension and different genes in solution could be added to each well and electroplated with an electroporation array. Such a system has been described in U.S. Pat. No. 6,352,853 King. et al. The disadvantages compared to our invention are; a higher volume of costly gene or drug solution will be required, imaging all the cells at one time is difficult, large arrays with 3000 or more discrete experiment sites are difficult to design and use efficiently and it is not possible to study cells electroporated with different genes in one chamber.
A paper by Huang and Rubinsky (Huang and Rubinsky. 1999) describes an early version of the concept of single or few cell electroporation with low voltages. The device described is different from our invention in that there is only one electrode site. The cell to be electroporated flows in solution until it is trapped in a hole and electroporated.
A paper by Lin and Huang (Lin and Huang, 2001) demonstrates that low voltage electroporation of cells plated on electrodes can be performed. Unlike our invention, the “microchips” described in this paper are not the type that can be manufactured by the thousands for affordability using standard silicon wafer fabrication technologies. Furthermore, each chip contains only one or a few independent electrodes. Our device can be manufactured of silicon using standard methods. Our initial device uses on chip multiplexers to control 3200 independent electrode sites, much larger numbers of sites are possible. This allows for low cost, one-time use and thousands of experiments to be performed in parallel. Another paper by the same research group (Lin et al., 2001) is similar to the above paper. It describes a flow through version of the same device. Cells in solution are passed between two electrodes for electroporation. The device is different from our invention in that the cells are not stationary and there is only one electrode.
There are a number of electroporation devices that involve fluidics or fluid flow management. In the case of our invention, the fluid containing a drug or gene flows but the cells are fixed, other inventions are designed to have the cells suspended in the fluid (U.S. Pat. Nos. 5,676,646, 5,545,130, 5,507,724, 5,704,908, 6,027,488, 6,074,605, 6,090,617 6,485,961). Some of these devices are for in vivo use to treat blood cells with genes or drugs. The blood flows through the device or the device is inserted into the blood vessel. Some of these devices are used to pump fluid into non-vascular body cavities. Some of these devices are designed for flowing a cell suspension through an electroporation chamber. None of the above devices are for flowing different solutions carrying different genes, drugs etc for electroporation of stationary cells.
U.S. Pat. No. 6,393,327 (Scribner) describes the stimulation array used in the present invention. The previous patent does not describe the electroporation application, processes or additional technology required.
The voltages required to electroporate cells depend on the distance between the electrodes. A paper by Nolkrantz demonstrates a single cell, DC voltage electroporation system with required voltages to distance ratios (electric fields) of 137 V/cm with a 30 μm diameter source electrode and saline bath return electrode. They calculated a cellular transmembrane voltage of 206 mV for 5-second pulses. In general the lower the voltage to distance ratio the longer the required pulse duration. The reported transmembrane voltages required for 1 ms pulses are 200-300 mV (Ryttse et al), equivalent to 133-200 V/cm. For the present invention the absolute voltages required at the electrode surface would be less than 3V. The distance between adjacent unit cells (center-to-center) is 30 and 50 μm in the x and y directions. That would yield a voltage to distance ratio of at least 600V/cm or 1000 V/cm, more than sufficient for electroporation with 5 sec or 1 ms pulses durations. In the Nolkrantz report, the spread of electroporation was limited to the single cell being electroporated.